The invention relates generally to the field of chemical analysis concerned with identification of organic compounds in complex mixtures.
Liquid chromatography is a widely used separation process which relies on the differential adsorption properties of organic molecules. Typically an organic mixture in a specific solvent is added to the top of a tubular column which has been packed with a fixed bed of adsorbent material providing surface area onto which substances may be adsorbed. As the solvent and solute mixture descend through the column, more strongly adsorbed compounds coat the packed bed surfaces, referred to as the stationary phase. The less strongly adsorbed substances, proceed through the column, along with the solvent. Ideally, the substances are progressively retarded into well separated segments. The eluted separated components of the mixture are discharged from the other end of the column along with solvent or eluent. Properly separated, the organic compounds come out of the column at intervals spaced by relatively pure solvent effluent.
For high performance liquid chromatography (HPLC), narrow columns known as microbore columns, may be employed to reduce solvent consumption and promote high solute concentrations. A commercially available microbore HPLC column 50 cm long with a 1 mm internal diameter is loaded with 10 micrometer (um) silica beads. In normal phase chromatography, hydrocarbon solvents such as hexane and dichlormethane are used in the mobile phase. In reverse phase chromatography, polar organic solvents such as methanol are used in combination with water.
Once separated by chromatography, the individualized organic substances can be analyzed for identification by a variety of techniques, including, for example, infrared (IR) spectroscopy, mass spectrometry, nuclear magnetic resonance, differential refractometry, heat-of-absorption detection and modified hydrogen flame ionization detection. In particular, the high scan speed and sensitivity of Fourier transform infrared (FTIR) spectroscopy has greatly facilitated the recording of characteristic infrared spectra of the individual components of mixtures separated by chromatographic techniques. Organic molecules in general contain interatomic bonds which exhibit characteristic resonance frequencies, many of which happen to be in the mid-IR region. These can be identified in the absorption spectrum of the material by observing the portion of radiation not absorbed but transmitted or reflected from the substance. Indeed, FTIR spectroscopy has already proved highly successful for gas chromatography (GC) as opposed to liquid chromatography.
Gas chromatography is a process by which complex mixtures of chemical compounds are separated from one another by selective partition between a stationary liquid or solid phase and a mobil gas phase, typically employing hydrogen, helium, nitrogen or argon. Although simpler to interface with FTIR spectroscopy because these mobile phases do not absorb infrared radiation, gas chromatography is only useful for lighter more volatile compounds. Many high molecular weight compounds of interest in fossil fuel studies, for example, are beyond the range of gas chromatography because they are not sufficiently volatile for GC separation. Moreover, the sensitivity of the popular light pipe method of GC/FTIR is reduced for less volatile compounds due to high light pipe temperatures which reduces infrared transmission of the interface.
Interfacing HPLC with FTIR is hampered by infrared absorption of the extraneous solvent remaining in the mobile phase after separation. Two types of interfaces have appeared in the literature: (1) flow cells, which allow recording the IR spectra while the HPLC effluent flows by a window transparent in the infrared and (2) solvent deposition systems which involve transfer and elimination of the solvent on a medium compatible with infrared sampling.
In flow cells, the spectral contribution including spectral masking produced by the solvent material, which is still present at full strength, must be taken into account. Thus, analysis by the flow cell method is limited to solvents which happen to be transparent in wide regions of the infrared spectrum and even then, some areas of the spectrum will remain opaque resulting in loss of information and sensitivity.
Solvent deposition designs, on the other hand, have utilized diffuse reflectance (DR) FTIR and transmittance spectroscopy. The DR/FTIR interface involves the depositing of concentrated portions of HPLC effluent into a series of mesh-bottomed diffuse reflectance cups containing potassium bromide (KBr) powder, practically the only suitable material transparent over the entire intermediate infrared range examined in IR spectroscopy. Unfortunately, because KBr is water soluble, it is incompatible with reverse phase chromatography. In addition, the cups themselves and the manner of sequencing them under the discharge end of the column give rise to relatively complex mechanical designs. After the solvent (normal phase) has evaporated from a given cup, the cup is brought into the beam path of the spectrometer and the diffuse reflectance spectrum is recorded. Although this system has demonstrated good sensitivity, especially with microbore HPLC columns, it does not allow continuous analysis of the chromatographic effluent because the cups take discrete samples. This is a considerable disadvantage because a component can be missed or more than one component may collect in a single cup. Furthermore, continuous recording of the spectra offers advantages in the presentation and interpretation of the spectral data, such as Gram-Schmidt reconstructions, wavelength chromatograms or the plotting of successive spectra to identify closely eluting compounds.
Continuous collection with a DR/FTIR interface has been accomplished recently using super critical fluid (SCF) chromatography. The deposition of the sample onto a bed of KBr powder was facilitated by the easy elimination of the mobile phase (CO.sub.2) which is a gas at atmospheric pressure. However, this particular principle does not appear to be applicable to conventional HPLC using liquid phases.
Transmission spectroscopy, as opposed to DR spectroscopy, is utilized in another solvent deposition system in which the effluent from a microbore HPLC flows directly onto a moving rectangular KBr crystal. After evaporation of the solvent, the crystal is passed through the spectrometer beam and transmission spectra are collected, thus providing a continuous spectral record of the chromatographic experiment.
In the past, the extension of either flow cell or solvent deposition interfaces to reverse phase chromatography employing aqueous solvents has required a solvent change before analysis. Unfortunately, the sensitivity of the technique is thereby reduced by at least an order of magnitude.
Laboratories involved in qualitative and quantitative analysis in chemical, biological, clinical and environmental research in general require more practical, reliable and versatile methods for analysis of compounds separated by HPLC.